Method for the inactivation of pathogens in donor blood, blood plasma or erythrocyte concentrates in flexible containers under agitation

ABSTRACT

The invention relates to a method for the inactivation of pathogens such as bacteria and viruses in donor blood, blood plasma and erythrocyte concentrates by photodynamic treatment and/or irradiation with ultraviolet light in flexible irradiation bags under intense movement.

The invention relates to a method for inactivating pathogens such asbacteria and viruses in donor blood (blood), blood plasma (plasma)and/or erythrocyte concentrates (ECs) by photodynamic treatment and/orirradiation with ultraviolet light.

It is known that therapeutic use of blood and blood preparations entailsthe risk of infecting the recipients with viruses and bacteria. Examplesinclude viral hepatitis B (HBV) and hepatitis C (HCV) as well as theAIDS pathogens HIV-1 and HIV-2. The risk always exists when no step istaken for inactivation and/or elimination of pathogens during theproduction of such preparations.

There have been a number of efforts to decontaminate blood preparationsby photodynamic methods. The principle is based on light treatment ofthe respective product in the presence of a photoactive substance (aphotosensitizer). The incident light must include a wavelength rangewhich is absorbed by the photosensitizer and by which it can beactivated. The absorbed energy is either transferred directly to therespective target structure (e.g. the nucleic acid or surface proteinsof a virus), which is thereby destroyed, or is transferred to dissolvedoxygen molecules, which are thereby activated. This results in formationof singlet oxygen, which has a strong virucidal and bactericidalactivity.

The photosensitizer used ideally has a high affinity for essentialcomponents of viruses and other pathogens, e.g. for their nucleic acids,and little or no affinity for the constituents of a preparation that isto be decontaminated. As a result of the photodynamic treatment, in thiscase the pathogens are inactivated while the product activity isretained. For example, methylene blue, a phenothiazine dye, has beendescribed as a suitable photosensitizer for treatment of plasma.Riboflavin (vitamin B2) is used for decontamination of plateletconcentrates, and phthalocyanines have been tested for decontaminationof ECs. However, methods for photodynamic inactivation of pathogens inECs have not yet gone beyond the laboratory scale.

This is true to an even greater extent for blood itself. One of the mainreasons for this is to be sought in the fact that the incident lightmust have a certain intensity to be able to activate the photosensitizerused and the blood and ECs have a very low permeability for light ofthat wavelength. This problem of course also occurs with plasma,although not to the same extent.

It is also known that it is possible to inactivate pathogens by simplyirradiating specimens with ultraviolet (UV) light of a short wavelength,i.e. in the wavelength range between approximately 200 nm and 320 nm, inparticular 200 nm to less than 300 nm (UVB and UVC). Above 320 nm, theenergy of the radiation is too low to inactivate microorganisms andviruses. In comparison with chemical, photochemical and photodynamicmethods of pathogen inactivation, mere irradiation with UV lightessentially has the advantage of being effective for itself alone andnot requiring the addition of reactive chemicals or photoactivesubstances.

UVC is the most effective for direct inactivation of pathogens. However,it has the disadvantage that it can penetrate through solutionscontaining proteins such as plasma and/or cloudy suspensions (e.g. bloodand ECs) only to a very low depth of penetration. UVC was used duringthe Second World War and shortly thereafter for sterilizing plasma andalbumin solutions, especially to inactivate hepatitis viruses. At thattime, the procedure was to pass a solution as a thin film in aflow-through apparatus past a UVC light source. This method did notprove to be reliable enough and was given up (N. R. Kallenbach, P. A.Cornelius, D. Negus, et al. Inactivation of viruses by ultravioletlight. Curr. Stud. Hematol Blood Transfus. 1989, 56, 70-82).

Methods that operate according to the same principle but have beendeveloped further are being used today for sterilizing therapeuticplasma protein preparations. In all cases, the aim is to treat largervolumes, i.e. plasma pools and/or protein solutions of a few hundredliters or even more (H. Hart, K. Reid, W. Hart. Inactivation of virusesduring ultraviolet light treatment of human intravenous immunoglobulinand albumin. Vox Sang 1993; 64(2):82-88 and S. Chin, B. Williams, P.Gottlieb, et al. Virucidal short wavelength ultraviolet light treatmentof plasma and factor VIII concentrate: protection of proteins byantioxidants; Blood 1995; 86(11):4331-4336).

The aforementioned flow-through apparatuses are not suitable forsterilizing multiple individual units from blood donors, plasma or ECshaving volumes of up to a few hundred mL. However, this is preciselywhat is needed in everyday practice at a blood bank.

UVB is both microbiocidal and virucidal, although not to the same extentas UVC. It penetrates into solutions containing protein and cloudysuspensions somewhat better than UVC, but its depth of penetration inplasma, for example, is in the range of only a few millimeters.

Plasma and ECs are usually isolated from individual blood donations butare also obtained from individual donors by machine apheresis. Thevolume of the preparations is generally between approximately 200 and350 mL. The volume of blood donations is usually between 450 and 500 mL.The preparations are either deep frozen (plasma) in flat plastic bags orare stored at approximately 4° C. (blood donations, ECs).

It would be desirable to sterilize these preparations in such bags butthe problem encountered here, as already mentioned, is that they arevirtually impermeable for UV light, and blood and ECs are alsoimpermeable for visible light.

It has surprisingly been found that the above problem is solved by amethod according to Claim 1. Preferred embodiments are the subject ofthe dependent claims or are described below.

According to the present invention, the preparations, i.e. donor blood(blood), blood plasma (plasma) and/or erythrocyte concentrates (ECs) aremoved in a suitable manner in their irradiation bags, so there isconstant turnover of the samples in the container. The movement is sointense that the layers which develop in areas within the liquid and/orsuspension are so thin that the light that is used can penetrate throughthem. At the same time the movement must be such that the liquid and/orsuspension is effectively mixed in the bag. Both are achieved if thefollowing prerequisites are met:

-   -   1. The irradiation bags are extremely flexible and they are not        secured during the light treatment, e.g. clamped between glass        plates or quartz plates. Therefore, they adapt to the change in        shape of the plasma and/or suspension (blood, ECs) that results        when the bags are in motion.    -   2. The irradiation bags are filled to max. 30%, in particular        max. 15% of the maximum filling volume.    -   3. The bags are moved vigorously, e.g. either horizontally        (linearly in a back and forth movement and/or in a circle or        ellipse) or vertically (rocking movement).    -   The term “vigorous movement” as used here is understood to refer        to the following (either individually or jointly):        -   1. It goes beyond a mere movement which causes mixing of the            liquids and/or suspensions.        -   2. Within the liquids and/or suspensions that are moved            vigorously, areas so thin that that they allow the            penetration of UV light and/or visible light (the latter is            true of cloudy and/or pigmented liquids and/or suspensions,            e.g. ECs) are formed at least temporarily, even in different            locations.        -   3. The reversal of the direction of vigorous movement is so            abrupt that most of the preparation that is in the            irradiation bag moves further in the original direction due            to its inertia and thus the remainder can form a thin layer            through which the type of light that is used can penetrate.

In conjunction with the constant mixing that takes place at the sametime, ultimately the entire preparation (and the pathogens containedtherein) is treated with light and thus sterilized.

The irradiation bag is only a few mm thick in the horizontal filledcondition, e.g. less than 10 mm, preferably less than 5 mm, and isintended to hold sample volumes of 200 mL to 500 mL, for example. Themaximum capacity (volume) of the irradiation bag is greater by a factorof at least 3, usually at least 6.66 times larger, preferably at least10 or even 20 times larger than the actual volume of specimen containedtherein to be treated.

Experimental Investigations

The experiments described here illustrate the efficacy of the method andare not limited to inactivation of the viruses mentioned. There are alsono restrictions with regard to the plasma and/or ECs originating fromblood donations used in the experiments described here. The inventivemethod may also be applied to preparations produced in some other way.All the experiments described here were performed three to six times.The results reported in each case represent the averages of theseexperiments.

Plasma Units and Erythrocyte Concentrates

The plasma units used and the ECs were prepared from individual blooddonations by the usual methods. They had a volume of approximately 250mL to 300 mL and up to 350 ml, respectively, and were stored in theusual plastic bags used for blood preparations. The remainingleukocytes, i.e. white blood cells, and/or platelets were removed byfiltration. The ECs were suspended in the stabilizer medium SAG-M. Theplasmas were stored at temperatures below −30° C. and were thawed in awater bath for the experiments. The ECs were stored at 4° C. to 8° C.under refrigeration.

Virological Tests

Plasma aliquots and/or EC aliquots were spiked with vesicular stomatitisvirus (VSV, Indiana strain, ATCC VR-158), sindbis virus (ATCC VR-68) andSuid herpes virus (SHV-1, pseudorabies virus, strain Aujeszky, ATCCVR-135), respectively. The virus titers were determined by means of CPEassay (CPE=cytopathic effect) and expressed as TCID₅₀ (TCID=tissueculture infective dose). Vero cells were used as the indicator cells.The initial virus concentration in the experiments performed wasapproximately 10⁵ to 10⁷ TCID₅₀.

Irradiation Devices, Irradiation Bags

One of the irradiation systems used was equipped with tubes that emitUVC light (wavelength: 254 nm). The specimens were irradiated from bothsides of the irradiation bag, i.e. from above and below. A secondirradiation device was equipped with tubes that emit UVB light (280-320nm). Irradiation was from both sides, too. A third irradiation devicewas equipped with LEDs (light-emitting diodes) that emit an intense redlight in the wavelength range around 635 nm. All three systems wereplaced on an orbital shaker during operation (manufacturer Bühler,Tübingen; model SM 25) that executed up to 100 revolutions per minute.The irradiation bags that were used were made of UV-permeable and highlyflexible plastic film.

EXPERIMENTAL EXAMPLE 1

Inactivation of VSV in Plasma by UVC: Influence of Agitation Speed andFree Mobility of the Plasma During Irradiation

Plasma units in irradiation bags were spiked with VSV and irradiated for2 minutes with UVC. A sample was agitated at 100 rpm and was clampedsecurely between two quartz plates during the light treatment. The othersamples simply lay on a quartz plate, so they could move within the bagduring the agitation. The rotational speed of the shaker was variedbetween 30 and 100 rpm. The results are summarized in Table 1. The virustiter in the fixedly clamped sample was reduced by a factor of onlyapproximately 0.3 log₁₀. TABLE 1 Shaking frequency Virus titer (rpm)(log₁₀ TCID₅₀) Comments 0 6.21 ± 0.69 Control 30 5.78 ± 0.27 Looselyplaced 50 4.59 ± 0.04 Loosely placed 75 0.92 ± 0.24 Loosely placed 1000.35 ± 0.52 Loosely placed 100 5.92 ± 0.11 ClampedWith the loosely placed samples, the rotational speed had a directinfluence on the extent of virus inactivation: at 30 and 50 rpm, theinactivation factors in comparison with the untreated control sampleswere only approximately 1.1 and 2.4 log₁₀, respectively, but at 75 rpmthey rose to 5.1 log₁₀ and at 100 rpm they rose to approximately 6.6log₁₀. The results of this experiment prove that plasma must be shakenthoroughly during the treatment in order for the irradiation with UVlight to be effective. However, in order for the shaking effect to alsobe effective, the samples must be placed loosely so that thin layersthrough which the light can pass are formed during the shaking.

EXPERIMENTAL EXAMPLE 2

Inactivation of VSV, Sindbis Viruses and SHV-1 in Plasma by Irradiationwith UVC: Inactivation Kinetics

Plasma units were spiked with VSV, sindbis viruses or SHV-1 andirradiated for 1-5 minutes. Samples placed loosely on the orbital shakerwere moved at 100 rpm. Control samples were irradiated for 5 minutes butnot shaken. Table 2 summarizes the results of the experiments. The VSVtiter in the shaken samples was reduced by a factor of more than 6.5log₁₀ within 3 minutes, whereas the inactivation factor in the unshakencontrol sample did not exceed 1.5 log₁₀. Sindbis viruses proved to bemore stable than VSV, but the great difference between shaken andunshaken samples was manifested here again. After an irradiation time of5 minutes, the virus titer in the shaken sample had decreased byapproximately 5.1 log₁₀ but the titer in the unshaken sample haddecreased by only 0.3 log₁₀. A similar result was obtained when SHV-1was used: in the shaken samples, the virus titer was reduced by a factorof 4.3 to 4.5 log₁₀ within 4 to 5 minutes; in the unshaken samples, itwas reduced by only 0.3 log₁₀ after 5 minutes of irradiation. TABLE 2Virus titer (log₁₀ TCID₅₀) UVC (min) Shaken VSV Sinbis SHV-1 Control −6.74 ± 0.32 7.01 ± 0.24 4.95 ± 0.23 2 + 0.95 ± 0.31 4.68 ± 0.21 2.56 ±0.25 3 + ≦0.24 ± 0.00  3.27 ± 0.16 1.67 ± 0.37 4 + ≦0.24 ± 0.00  2.10 ±0.12 0.66 ± 0.29 5 + ≦0.24 ± 0.00  1.86 ± 0.09 0.42 ± 0.21 5 − 5.69 ±0.18 6.73 ± 0.16 4.65 ± 0.16

EXPERIMENTAL EXPERIMENT 3

Inactivation of VSV in Plasma by Irradiation With UVB: InactivationKinetics

Plasma units were spiked with VSV and irradiated from 1 to 5 minutes.The samples placed loosely on the orbital shaker were moved at 100 rpm.A control sample was irradiated for 5 minutes but was not shaken. AsTable 3 shows, the virus titer in the shaken samples was reduced by afactor of 6.36 log₁₀ within 5 minutes but the titer in the unshakencontrol sample was reduced by only approximately 1.5 log₁₀. Theseresults show that the phenomenon discovered—the increase in pathogeninactivation in loosely placed samples due to intense shaking—is notlimited to UVC. TABLE 3 Virus titer UVB (min) Shaken (log₁₀ TCID₅₀)Control − 7.00 ± 016  2 + 4.70 ± 0.08 3 + 3.68 ± 0.12 4 + 2.23 ± 0.235 + 0.64 ± 0.08 5 − 5.52 ± 0.08

EXPERIMENTAL EXPERIMENT 4

Inactivation of VSV in Plasma by Photodynamic Treatment With MethyleneBlue and Light

Plasma units were spiked with VSV, mixed with 0.25 μM/L of thephotosensitizer methylene blue (MB) and irradiated with red LED light onan orbital shaker at a rotational speed of 100 rpm for up to 30 minutes.Control samples were treated for 20 minutes in the presence of the sameconcentration of MB, but were not shaken during treatment.

As Table 4 shows, the extent of virus inactivation in the shaken sampleswas much greater than that in the unshaken samples. In the latter, thevirus titer had dropped by a factor of approximately 4.4 log₁₀ after 20minutes and by approximately 5.8 log₁₀ after 30 minutes. In the unshakensamples, the reduction factor after 20 minutes was no higher thanapproximately 2.7 log₁₀. TABLE 4 MB/light Virus titer (min) Shaken(log₁₀ TCID₅₀) Control − 6.72 ± 0.24 10 + 4.95 ± 0.68 20 + 2.30 ± 0.8830 + 0.94 ± 0.87 20 − 4.04 ± 0.54

EXPERIMENTAL EXAMPLE 5

Inactivation of VSV in ECs by Photodynamic Treatment With Methylene Blueand Light

EC aliquots were spiked with VSV, mixed with 5 μM/L of thephotosensitizer methylene blue (MB) and irradiated with red LED light onan orbital shaker at a rotational speed of 100 rpm for up to 30 minutes.However, the control samples were not moved during light treatment.Table 5 shows the clear-cut results of this experiment. It is obvioushere that virus inactivation proceeded much more rapidly in the shakenEC samples than in the unshaken samples. In the samples that were shakenduring treatment, the virus was almost completely inactivated after 30minutes (inactivation factor 6.7 log₁₀). In contrast, the reductionfactor in the unshaken samples was only approximately 2.7 log₁₀ after 30minutes.

The results of Experimental Examples 4 and 5 prove that also theefficacy of the photodynamic treatment of plasma or erythrocyteconcentrates is increased enormously if the samples are shakenvirgorously during light treatment. TABLE 5 MB/Light Virus titer (min)Shaken (Log₁₀ TCID₅₀) Control − 7.04 ± 0.26 10 + 2.62 ± 0.31 20 + 0.89 ±0.21 30 + 0.30 ± 0.12 10 − 5.07 ± 0.26 20 − 5.25 ± 0.31 30 − 4.35 ± 0.27

1. A method for inactivating pathogens in donor blood, blood plasmaand/or erythrocyte concentrates, comprising the following steps:preparing blood donations and/or preparations obtained from donor bloodand/or by machine apheresis, (a) adding a suitable photoactive substanceand photodynamic treatment by irradiation with light, comprising orconsisting exclusively of wavelengths in the absorption range of thephotoactive substance, or (b) irradiating the preparations withultraviolet (UV) light at wavelengths of 200 nm to 320 nm, wherein thepreparations consist of numerous units that can be handled individuallyand stored separately, and the preparations are in flat, flexiblelight-permeable (alternative (a)) and/or UV-permeable (alternative (b))irradiation bags, characterized in that the irradiation bags are filledto less than 30 vol % of the maximum filling volume of the irradiationbags, and the irradiation bags are shaken during the photodynamictreatment and/or irradiation with UV light, so that the contents of theirradiation bags are circulated and zones of variable layer thicknessdevelop due to this movement.
 2. The method according to claim 1,characterized in that the pathogens are viruses and/or bacteria.
 3. Themethod according to claim 1, characterized in that due to the movement,the zones have regions which regularly have layer thicknesses oftemporarily less than 1 mm, preferably less than 0.05 mm.
 4. The methodaccording to claim 1, characterized in that the movement, in particularthe amplitude of the movement, takes place in such a way that regionsdevelop within the preparations in which the layer thickness isregularly temporarily less than 0.05 mm.
 5. The method according toclaim 1, characterized in that the sum of the areas of the bottom sideand the top side of the irradiation bags that are/can be in contact withthe contents of the bag amounts to more than 90% by area, preferablymore than 99% by area, of the total internal surface area of the bagcontents.
 6. The method according to claim 1, characterized in that inthe case of embodiment (b), the irradiation is or includes UVB of lessthan 320 nm to 280 nm and/or UVC of less than 280 nm to 200 nm, inparticular UVC of less than 260 nm to 220 nm and preferably consistsexclusively of radiation with wavelengths in the ranges given above. 7.The method according to claim 1, characterized in that each unitoriginates from one to six donors, preferably one donor.
 8. The methodaccording to claim 1, characterized in that the irradiation is with UVBhaving a light energy of 0.3 J/cm² to 10 J/cm², preferably 0.5 to 5J/cm².
 9. The method according to claim 1, characterized in that theirradiation is with UVC having a light energy of 0.01 to 5 J/cm², inparticular 0.1 to 2 J/cm².
 10. The method according to claim 1,characterized in that the irradiation bags have a volume of up to 5000mL.
 11. The method according to claim 1, characterized in that theirradiation bags are held movably in the apparatus in which they aremoved and irradiated, and in particular they are not clamped between twosurfaces.
 12. The method according to claim 1, characterized in that theirradiation bags are moved during at least three-quarters of the totalirradiation time, in particular at least five-sixths of that time. 13.The method according to claim 1, characterized in that the irradiationbags are moved by shaking, tilting and/or rotating.
 14. The methodaccording to claim 1, characterized in that the preparations are plasmaand consist of more than 80% by weight blood plasma.
 15. The methodaccording to claim 1, wherein the preparations are erythrocytepreparations and have a hematocrit between 10% and 75% by weight,preferably between 20% and 60% by weight.
 16. The method according toclaim 1, characterized in that the photoactive substances include one ormore phenothiazine dyes, in particular thionine, methylene blue,toluidine blue and/or the azure dyes A, B and C.
 17. The methodaccording to claim 1, characterized in that the photoactive substanceincludes one or more phthalocyanine compounds.
 18. The method accordingto claim 1, characterized in that the photoactive substance comprisesone or more phorphyrin compounds.
 19. The method according to claim 16,characterized in that the photodynamic treatment is performedexclusively with wavelengths in the absorption range (±20 nm) of thephotosensitizer(s) used.
 20. The method according to claim 1,characterized in that the irradiation bags are filled to max. 15% of themaximum filling volume.
 21. The method according to claim 1,characterized in that blood plasma is treated according to step (a) andin the absence of a photosensitizer.
 22. The method according to claim1, characterized in that the shaking is performed with an orbitalshaker, a platform shaker, a rocking shaker or a tumbling shaker. 23.The method according to claim 1, characterized in that the irradiationbags are placed on one side, so that the height of the irradiation bags,based on the distance (surface normal) between the surface on which theirradiation bags are lying and the point of intersection with the uppersurface of the irradiation bag, changes constantly during and due to themovement, or shaking, when viewed over the total upper surface of theirradiation bag.
 24. The method according to claim 1, characterized inthat the irradiation bag has an average filling height of less than 5 mmand wave valleys having layer thicknesses of less than half the averagefilling height, preferably layer thicknesses of less than 1 mm or evenless than 0.05 mm are produced constantly due to the movement.
 25. Themethod according to claim 1, characterized in that the irradiation bagsare moved constantly with an amplitude of more than 0.2 mm, preferablymore than 1 cm and especially 1 to 15 cm or 2 to 8 cm at least in the xdirection and optionally also in the y direction (y direction at a rightangle to x direction) during irradiation, and regardless thereof thefrequency of the change in direction of movement is greater than 0.5 Hz,preferably 1 to 10 Hz.